Early region 4 (E4) of human adenoviruses is located at the right end of the viral genome. It encodes seven proteins, four of which have been implicated in regulatory processes in infected cells. The goal of the studies proposed here is to determine the mechanisms of action of three E4 products, those of open reading frames (ORFs) 3, 4, and 6. ORFs 3 and 6 are required for the expression of viral late genes and act at a post-transcriptional step in mRNA synthesis: mutants lacking the products of these genes transcribe the late region normally, but fail to accumulate late mRNA or its nuclear precursors. The proteins are functionally redundant, and their functions in this capacity are specific for viral RNA. Studies of the activities of these proteins will be threefold. First, it will be determined, using antibodies specific for the ORF 3 and 6 products, whether those products associate in infected cells with viral RNA or with structures known to be involved in gene expression (such as snRNPs). Second, the proteins will be assayed for the ability to bind to viral late RNA in vitro. Finally, the phenotype of ORF 3-,ORF 6- mutants will be duplicated in an in vitro system to permit its biochemical dissection. The E4 ORF 4 product negatively regulates viral DNA replication: an E4 mutant that produces only the ORF 4 product is profoundly deficient in viral DNA replication, while mutants lacking all of E4 produce DNA normally. Mutants expressing ORF 4 also inhibit DNA synthesis by other E4 deletion mutants in trans. To determine whether the ORF 4 protein inhibits replication via an effect on the synthesis of viral replication proteins, their expression in E4 mutant infections will be examined. If alterations of the expression of replication proteins cannot account for the ORF 4 effect, direct interactions between the ORF 4 product and the replication machinery will be probed using ORF 4-specific antibodies. Finally, extracts from mutant-infected cells will be assayed for the ability to synthesize viral DNA, and if deficient, will be used to identify the defective or missing components.